The acid-sensing capability of the pH-Low Insertion Peptide (pHLIP) makes it a potential tool for exploiting the extracellular acidity associated with pathological conditions such as cancer, inflammation, and ischemia. How- ever, because the relationship between cell membrane composition and pHLIP and the apparent pK of pHLIP insertion is intuitive at best, a fundamental gap exists in our understanding of the biophysical proteolipid interac- tions that govern binding, folding, and insertion of this peptide. The long-term goal is to develop approaches that facilitate efficient design of membrane-insertion peptides for use in targeted delivery of pharmaceuticals and imaging agents. The overall objective is to identify the molecular interactions between pHLIP and the cell mem- brane that lead to binding and insertion and to characterize the associated thermodynamics of this process. The central hypothesis is that the effectiveness and timescales of pHLIP binding and insertion are directly dependent on the composition of the peptide and the lipid bilayer and the free energy landscape of the cell membrane. The rationale for the proposed research is that knowledge of the molecular interactions and thermodynamics that govern pHLIP binding and insertion will lead to a more comprehensive understanding of how pHLIP functions as well as initiating development of theoretical approaches that allow for efficient rational design of pHLIP variants with tunable properties such as the apparent pK of insertion. This hypothesis will be tested by pursuing three specific aims: 1) Determine the effect of bilayer surface electrostatics on pHLIP binding; 2) Determine the order of protonation during pHLIP insertion; and 3) Identify the intermediate states of pHLIP insertion. Under the first aim, an already proven enhanced sampling molecular dynamics (MD) simulation approach, which has been established as feasible in the applicant?s hands, will be used to model binding of pHLIP to a lipid bilayer surface. Conserved binding motifs and thermodynamics of pHLIP binding will be analyzed for bilayers with a range of surface electrostatic properties. Under the second aim, enhanced sampling MD simulations will be used to model insertion of pHLIP into a lipid bilayer. A range of protonation states of pHLIP will be tested, allowing for determi- nation of the cooperative change in thermodynamics during insertion that is associated with protonation of ani- onic residues. Under the third aim, enhanced sampling MD simulations will be used to explore the exit of pHLIP from the cell membrane. pHLIP variants with different entry/exit kinetics will be investigated to determine the cumulative effect of acidic residues on the exit process. The approach is innovative, because it represents a new and substantive departure from the status quo by shifting focus to modeling-based high-throughput optimization of pHLIP peptide function. The proposed research is significant, because it is expected to have widespread importance in the utilization of membrane-insertion peptides as drug delivery agents in the treatment of carcino- genic and inflammatory disorders. Ultimately, such fundamental knowledge has the potential to inform the de- velopment of pHLIP variants with tunable pH-sensing capabilities and facilitate the aforementioned applications.